MICROBIOLOGY CULTURE INSTRUCTIONS                                                                                 Microbiology Programs

Inspect the fiberboard container carefully for all inclusions. After inventorying the contents of the container, store them in a refrigerator. These instructions are illustrated in the Program Guide online under Microbiology Instructions.

Special Safety Precautions - These specimens contain pathogens or potential pathogens and should be considered infectious and handled as though they are capable of transmitting disease. They should be handled and disposed of only by personnel trained to work with pathogenic bacteria. All laboratory precautions and safety measures appropriate to handling live cultures should be practiced when working with these specimens.

In addition to the Precautions section of the program guide, be especially careful to avoid aerosol creation, inhalation, ingestion or injection of bacteria. These specimens should be disposed of as hazardous waste.

Rehydration Instructions for Swab Samples – These specimens are stabilized viable microorganisms lyophilized directly to an inoculation swab. A Rehydration Fluid tube is provided for each swab. The Microbiology Rehydration Fluid is located with the rest of your samples and is not found inside the tube with the swabs. The Diluent vials are placed in a pouch labeled REHYDRATION FLUID. There are up to 5 equivalent vials, each containing approximately 0.5 mL of TSB. The vials are not specifically labeled for any sample or Microbiology program module. We recommend you label the rehydrating vial prior to use to avoid confusion.

a. Warm appropriate amount of media, one specimen swab and one vial of rehydrating fluid to room temperature (20-25ºC) for each specimen tested. Store the second swab and extra rehydrating fluid vials in the refrigerator in case repeat testing is necessary.

b. Remove swab from plastic tube and submerge the sample portion into the fluid. Allow approximately 10 seconds for the lyophilized specimen to liquify while mixing and swirling the swab gently.

c. Once the swab is saturated, inoculate your media directly with the swab. Return the swab to the rehydration fluid before inoculating each subsequent culture plate.

d. If you perform a gram stain as part of your identification procedure, you can now transfer a portion of the liquified specimen to a clean glass slide. Be sure to have inoculated all culture media prior to using the specimen swab to prepare your gram stain. NOTE: There will be a small amount of charcoal residue from the lyophilization process present on your gram stain slide. This should not interfere with your ability to determine the staining pattern of the organism.

e. Continue by following the procedure and methods used by your lab to identify the organism(s) present in these specimens.

For Uricult users or users of other tests that require a liquid sample.

a. Open each foil pouch at the tear slit and remove the swab from the foil pouch.

b. Submerge the swab portion only into the provided rehydration broth tube for 30 seconds to wet the swab.

c. Express as much material from the swab as possible by firmly pressing the head of the swab against the side of the rehydration broth tube.

d. Swirl the rehydration broth tube gently to ensure even mixing and then use the broth as you would a liquid sample.

Urine Colony Count (CC-) Samples – Each sample consists of lyophilized pellet in a labeled foil pouch and a labeled 99 mL bottle of dilution fluid.

a. Warm an appropriate amount of media, specimen pellet and 99 mL bottle of dilution fluid to room temperature (20-25ºC) for each specimen tested.

b. Remove the cap from the sample vial and open the flip-top on the dilution fluid bottle.

c. Empty the lyophilized pellet into the dilution fluid and recap the bottle securely. Be careful to avoid touching the pellet to avoid contamination.

d. Mix the contents of the bottle vigorously until the entire pellet has dissolved and the suspension is homogeneous in appearance.

e.The entire contents of the dilution bottle (which now contains the dissolved lyophilized pellet) simulates a urine sample.

  Proceed to test as you would a patient sample in your laboratory.

f. Continue by following the procedures and methods used by your lab to identify the organism(s) present in these specimens and/or perform colony count testing.

g. If you perform a gram stain as part of your identification procedure, you may transfer a small amount of the mixed solution from the first CC specimen to a clean glass slide. Be sure to use sterile technique when transferring the solution. NOTE: There will be a small amount of charcoal residue from the lyophilization process present on your glass slide. This should not interfere with your ability to determine the staining pattern of the organism.

h. Record your colony count results for only the first two CC specimens.

Determining Type (Extent) of Laboratory Service - All participants, regardless of the extent of their laboratory practice, evaluate the same specimens. In order to be graded appropriately, you must report the extent of laboratory practice (Extent 0, 1, 2, 3, 4 or 5) for each specimen. Refer to the Program Guide online to determine your extent.

1. Subject each specimen to your protocol for each source as described at each specimen number on your reporting form.

2. Based on what you would report in the context of your specific laboratory practice, determine your extent for each specimen independently of each other, according to the definitions on the reporting form and clarified in the following table:

Regardless of the extent reported, we are required to grade the most definitive identification reported. This means, for example, that a species result takes precedence in grading over a genus, antigen or Gram stain result.

If unable to decide between extents for a given specimen, report the lowest extent, which applies, except Extent 0. In order to preserve the opportunity to get a zero on one specimen and still receive 80 percent overall, it is advisable to avoid using Extent 0 when Gram stain (Extent 1) or antigen screening (Extent 2) results are available for reporting. It is acceptable to challenge your Gram stain or antigen screening procedure(s) with our bacteriology specimens (without regard to specimen source information) in order to receive five specimen scores upon which to generate an overall procedure score for bacterial identifications. We are required to categorize participants who fail to report their extent(s) as Extent 5 and to grade accordingly.

Coding for Presumptive Culture Identification (or other screening methods) - Participants reporting presumptive identifications by culture must not use result codes 726 to 911. If performing isolations only with selective media, these participants might need to report code 948 (No pathogen isolated), but should not report either code 949 (No aerobic growth) or 951 (No aerobic or anaerobic growth).

When using a presumptive culture or other screening methods for identification, be certain to respond using result codes appropriate for the origin of the sample being tested; result codes >911. CMS is particularly concerned that laboratories performing limited testing report their results in such a way as to reflect knowledge of the limitations of their method. Therefore, do not use codes such as 948 (no pathogens found) if your laboratory does not screen for GC on throat cultures, Campylobacter on stool cultures and so on. You MUST select a code that accurately reflects the limitations of your testing or you will be scored as incorrect.

Coding Extent 3, 4 and 5 Results - For the first four Specimens in each event, participants using Extent 3, 4 or 5 may report only the organism(s), which they consider to be the significant pathogen(s) that is/are clearly responsible for the illness described, excluding immuno-compromised patients. CLIA directs that we require reporting of all organisms present for at least one sample which contains multiple organisms. The last Specimen in each event will contain multiple organisms and may contain multiple pathogens. If you report extents 3, 4 or 5, you must report all organisms present or you will be flagged for an incorrect response. The significant pathogen must be reported as organism #1 when multiple organisms are detected.

Code your answers using the Organism Result Code list on the reveres side of these instructions. Anti-microbial susceptibility tests (ASTs) are to be performed on the most significant pathogen in first specimen only, using the AST codes listed on the reverse side of these instructions. Per CMS requirements participants will be flagged for inappropriate selection of Antimicrobial Agents as listed in the current edition CLSI (NCCLS) guidelines and M100. Selection of an incorrect Antimicrobial will be scored incorrect.

Refer to your reporting form for specimen sources and other reporting requirements. If you do not see a particular Anti-microbial Agent listed, please attach a note indicating that you would like for us to add a code for that agent. Note: We will not add any new Anti-microbials until they are listed in the CLSI guidelines.

ORGANISM RESULT CODES

Please review codes. Changes have been made to the organism codes.

Instructions for Handling INOCU-SWAB II™

Instructions for Handling INOCU-PELLETS